To invade target organs, many viruses breach epithelial surfaces and then use a lympho-hematogenous route of dissemination through the regional lymph node (LN) and then the blood. Immunology and Virology textbooks teach us that the primary function of lymph nodes (LNs) is to serve as sites of lymphocyte priming. Different to this textbook view, we and others have recently shown that LNs are also sites where various components of the innate and adaptive immune system mount an antiviral response critical for restriction of pathogen spread. Using ectromelia virus (ECTV) as a model virus, we have recently found that inflammatory monocytes (iMo) are rapidly recruited to the popliteal draining lymph node (draining LN) following footpad infection. Recruitment of iMo to the draining LN requires TLR9 and its adapter MyD88 expressed in trans by other CD11c+ cells, probably migratory cells from the skin including Langerhans cells, CD103+ CD207+ dermal dendritic cells (DC), and/or CD103- CD207- dermal DC. Our data strongly indicate that iMo play a key role in virus containment because they - and not plasmacytoid DCs, as previously believed - are the major producers of Type I interferon (T1-IFN) when they become infected. Notably, uninfected iMo produce pro-inflammatory cytokines. The major objective of this proposal is to characterize the recruitment and function of this critical cell type in vivo. In Aim 1 we will use several genetically modified mice and a variety of methods determine: 1) Whether and which skin-derived CD11c+ cells increase their migration to the draining LN in response to ECTV infection. 2) Which of the skin CD11c+ cells are required for iMo recruitment. 3) Whether TLR9/MyD88 in skin CD11c+ cells is required for iMo recruitment to the draining LN. In Aim 2, we will look at the iMo response to viral infection in the draining LN. Specifically we will: 1) Identify changes in global gene expression in infected and uninfected iMo and determine how these are affected by key innate signaling pathways, 2) Determine the mechanisms of T1-IFN and pro-inflammatory cytokine expression and 3) Visualize the interaction of iMo with NK cells and CD8+ T cells in draining LNs. The successful completion of these Aims will outline the mechanisms whereby iMo respond to infection in the draining LN, a process that is required for effectively controlling the lympho-hematogenous dissemination of a highly pathogenic virus in its natural host. These findings will be broadly applicable to an array of pathogenic viruses that disseminate lympho-hematogenously.